The present invention relates generally to solid phase processing and, more particularly, to an apparatus for dispensing reagents and other fluids to a plurality of reaction sites for solid phase processing including solid phase synthesis of complex chemicals such as oligonucleotides and the like.
A variety of separative, synthetic, and enzymatic or otherwise catalytic processes use beds of particulate material with transport of reactants, reagents and products or eluants in solution through the bed. In addition, many reactions are known in which the products are separated by concentration in one of two or more phases. These processes include, among others, ion exchange chromatography, gel filtration, ion exclusion chromatography, affinity chromatography, separations based on hydrophobicity, purification based on hybridization, peptide synthesis, oligonucleotide synthesis, and polysaccharide synthesis including combinations of the last three. These processes may be carried out on a small scale for analytical purposes or process design, and are then often scaled up for preparative work. In nearly all examples the solid phase particulates are packed in a closed column with a porous frit on the lower end, an optional frit at the top, and with fluid-connections at both ends so that liquid can flow in either direction through the bed. To achieve efficiency and high resolution with solid phase supports, all volume elements of all fluids should flow through paths of identical composition and nearly identical length, and all particles in the bed should be exposed to the same succession of liquids under the same conditions.
In solid phase systems, some interaction occurs between the solutes running through the bed and the particles composing the bed. This interaction may be based on secondary forces (ionic, hydrophobic, or on immunochemical interactions, or base pairing) or primary valencies as when amino acids or nucleotides are added to a growing chain on the solid phase support, or when immobilized enzymes cleave substrates flowing through the bed, or when enzymes in solution react with substrates attached to the packing. In addition, solvents or reagents of successively differing composition which dissociate adsorbed or otherwise attached bound molecular species, or which cleave off protective groups, or compounds including polymers which have been synthesized on the support may be made to flow through the support. The dissociated or cleaved substances then are free to flow out of the bed in flowing liquid.
In particular, nucleic acid synthesis (generally referred to as xe2x80x9cDNA synthesisxe2x80x9d) is the process of constructing synthetic single-stranded oligonucleotide through linking of nucleotide, the basic building blocks for DNA. In an automated system, the various steps are carried out by a reagent delivery system which dispenses a number of chemical reagents in a predetermined sequence in a cycle into a synthesis reaction column containing the solid-phase support, according to instructions from the system controller or computer. After the desired number of cycles have been completed, the synthesized oligonucleotide is separated from the reaction column and collected in a vial. This step is generally referred to as xe2x80x9ccleavagexe2x80x9d. The oligonucleotide may further be subject to a step generally referred to as xe2x80x9cdeprotectionxe2x80x9d to complete isolation of the oligonucleotide. In a process for synthesizing polynucleotides on a solid support, the solid support traditionally consists of glass beads of controlled porosity (CPG) or, more generally, of particles of a functionalized inorganic or organic polymer.
The isolation of oligonucleotide involves the treatment of the solid bound oligonucleotide with a cleavage and/or deprotection reagent. Typically, this reagent is concentrated ammonia solution in water but can be other homogeneous or heterogeneous solutions of appropriate bases, alcohols and water. The cleavage and deprotection process is typically performed in two steps. The cleavage of the oligonucleotide is performed at room temperature for approximately one hour before decanting the mixture into a pressure-sealable vessel for extended higher temperature treatment to effect the removal of secondary protecting groups on the synthetic oligonucleotide. This two step process reduces the quantity of support related contaminants in the final isolated product.
The use of a single nozzle for delivering different reagents into a reaction site, well, or column is not feasible because the nozzle will need to be cleaned or flushed out between reagents to avoid contamination, resulting in a high cost and a low throughput. In one conventional chemical synthesis system, a plurality of reagent dispensing nozzles are arranged in a linear array, and the plate containing the reaction cell(s) or column(s) is moved under the linear array to receive reagents from the dispensing nozzles one at a time. The throughput remains low.
Another synthesis apparatus is disclosed in U.S. Pat. Nos. 5,814,700, 5,837,858, and 6,001,311 employing an array of nozzles. A transport mechanism aligns the reaction wells and selected nozzles for deposition of the liquid reagent into the selected reaction wells. Elaborate manipulation of the transport mechanism is used to dispense reagents from the various nozzles into the various reaction wells in sequence to provide simultaneous synthesis in the reaction wells. The throughput is still relatively low because each nozzle can dispense only one reagent.
Embodiments of the present invention are directed to an improved chemical synthesis apparatus for performing chemical synthesis such as nuclei acid synthesis in a plurality of reaction wells or cells in an efficient manner. The apparatus employs dispenser heads that each include a cluster of nozzles which are fluidicly coupled to a plurality of reagent sources for dispensing different reagents through the single dispenser head. Because each dispenser head is capable of dispensing a plurality of different reagents, the apparatus can perform simultaneous synthesis in a plurality of cells at a high throughput without complex and elaborate control of movement of the dispenser heads relative to the cells.
In accordance with an aspect of the present invention, a multi-channel reagent dispenser head for introducing a plurality of reagents into a reaction site comprises a dispenser head body having a dispensing end which is configured to dispense a plurality of reagents from a plurality of reagent sources. A group of nozzles include a plurality of reagent dispensing nozzles which are fluidicly coupled with the plurality of reagent sources. The group of nozzles are clustered to provide a plurality of nozzle outlets in the dispenser head body to introduce reagents from the plurality of reagent sources through the dispensing end of the dispenser head body into the reaction site.
In some embodiments, the plurality of reagent dispensing nozzles are each separately coupled with one of the plurality of reagent sources. The plurality of reagent dispensing nozzles may be separately coupled with reagent sources of building block elements such as bases A, C, G, T, and an activator such as tetrazole. Alternatively, the plurality of reagent dispensing nozzles may be separately coupled with reagent sources of acid deblock, oxidizers, and capping agents. The group of nozzles desirably include a wash nozzle which is fluidicly coupled with a source of wash solvent. The wash solvent may comprise acetonitrile. The group of nozzles desirably include a vacuum nozzle which is coupled to a vacuum source. The nozzle outlet of the vacuum nozzle may be disposed proximal of the nozzle outlets of the reagent dispensing nozzles. In specific embodiments, the dispensing end of the dispenser head body has a maximum dimension of about 9 mm. The nozzles each have an outer diameter of less than about {fraction (1/16)} inch.
In accordance with another aspect of the present invention, a multi-channel reagent dispenser apparatus for introducing a plurality of reagents into a plurality of reaction sites comprises a plurality of reagent sources, and a plurality of reagent dispensing nozzles each coupled with one of the plurality of reagent sources. A plurality of dispenser heads each include a dispenser head body having a dispensing end which is configured to dispense a plurality of reagents from the plurality of reagent sources. Each dispenser head body has therein a group of nozzles being clustered to provide a plurality of nozzle outlets in the dispenser head body to introduce reagents from the plurality of reagent sources through the dispensing end of the dispenser head body into one of the reaction sites. The group of nozzles include more than one reagent dispensing nozzle from the plurality of reagent dispensing nozzles.
In some embodiments, a plurality of reagent dispensing nozzle valves are each coupled with one of the reagent dispensing nozzles to control reagent flow from the reagent sources to the reagent dispensing nozzles. At least one wash nozzle is coupled with at least one source of wash solvent. The group of nozzles clustered in each dispenser head body include at least one wash nozzle. At least one wash nozzle valve is each coupled with one of the at least one wash nozzle to control wash solvent flow from the at least one source of wash solvent to the at least one wash nozzle. The group of nozzles clustered in each dispenser head body include a vacuum nozzle which is coupled to a vacuum source. A vacuum nozzle valve is coupled with the vacuum nozzle to control vacuum flow through the vacuum nozzle.
In some embodiments, the plurality of dispenser heads comprise at least one first dispenser head and at least one second dispenser head. Each first dispenser head has therein a cluster of first nozzles being coupled with a first set of the plurality of reagent sources. Each second dispenser head has therein a cluster of second nozzles being coupled with a second set of the plurality of reagent sources which are different from the first set of reagent sources. The first set of reagent sources may comprise building block elements such as bases A, C, G, T, and an activator such as tetrazole. The second set of reagent sources may comprise acid deblock, oxidizers, and capping agents. A first actuator is configured to move each of the at least one first dispenser head from one reaction site to another reaction site to introduce reagents from the first set of reagent sources into the reaction sites. A second actuator is configured to move each of the at least one second dispenser head from one reaction site to another reaction site to introduce reagents from the second set of reagent sources into the reaction sites. A controller is coupled with the first and second actuators to automatically control movements of the at least one first dispenser head and the at least one second dispenser head to introduce reagents from the first and second sets of reagent sources separately into the reaction sites.
In specific embodiments, a plurality of reagent dispensing nozzle valves are each coupled with one of the reagent dispensing nozzles. The controller is coupled with the reagent dispensing nozzle valves to control reagent flow from the reagent sources to the first nozzles in the at least one first dispenser head and to the second nozzles in the at least one second dispenser head. The dispensing end of each dispenser head body has a maximum dimension of about 9 mm. The plurality of dispenser heads comprise a plurality of first dispenser heads and a plurality of second dispenser heads. The first dispenser heads are spaced about 9 mm apart, and the second dispenser heads are spaced about 9 mm apart.
The plurality of reagent sources each are delivered to the reagent dispensing nozzles by pressurization with an inert gas. The reaction sites are evacuated under vacuum assist. The reaction sites are disposed in an array provided in a plurality of vacuum trays which are formed on a single block. The apparatus is disposed in an inert environment, such as nitrogen or argon.
In accordance with another aspect of the present invention, a method for introducing a plurality of reagents into a plurality of reaction sites comprises providing a plurality of reagent sources, a plurality of reagent dispensing nozzles each coupled with one of the plurality of reagent sources, and a plurality of dispenser heads. Each dispenser head includes a dispenser head body having a dispensing end. Each dispenser head body has therein a group of nozzles being clustered to provide a plurality of nozzle outlets in the dispenser head body. The group of nozzles include more than one reagent dispensing nozzle from the plurality of reagent dispensing nozzles. The method further comprises controlling flows of reagents from the plurality of reagent sources through the plurality of reagent dispensing nozzles to dispense a plurality of reagents via the group of nozzles clustered in each dispenser head body through the dispensing end of the dispenser head body into one of the reaction sites.
In some embodiments, the flows of reagents through each dispenser head body are controlled by operating a plurality of reagent dispensing valves each coupled with one of the reagent dispensing nozzles based on flow rates to dispense preset amounts of reagents via the group of nozzles clustered in each dispenser head body at preset times through the dispensing end of the dispenser head body into one of the reaction sites. The flows of reagents through each dispenser head body are controlled to provide one reagent at a time through the dispenser head body.
The method may further comprise providing at least one source of wash solvent and at least one wash nozzle coupled with the at least one source of wash solvent, wherein the group of nozzles clustered in each dispenser head body include at least one wash nozzle. The wash solvent is dispensed through the at least one wash nozzle in each dispenser head body at preset times. The method may further comprise providing a vacuum nozzle in the group of nozzles clustered in each dispenser head body, and drawing a vacuum through the vacuum nozzle in each dispenser head body between dispensing different reagents through the dispenser head body.
In some embodiments, the plurality of dispenser heads comprise at least one first dispenser head and at least one second dispenser head. Each first dispenser head has therein a cluster of first nozzles being coupled with a first set of the plurality of reagent sources. Each second dispenser head has therein a cluster of second nozzles being coupled with a second set of the plurality of reagent sources which are different from the first set of reagent sources. The method further comprises moving each of the at least one first dispenser head from one reaction site to another reaction site to introduce reagents from the first set of reagent sources into the reaction sites. The method may further comprise moving each of the at least one second dispenser head from one reaction site to another reaction site to introduce reagents from the second set of reagent sources into the reaction sites. The at least one first dispenser head and the at least one second dispenser head are moved automatically by computer control.
In specific embodiments, the plurality of dispenser heads comprise a plurality of first dispenser heads, and flows through the first nozzles in the first dispenser heads are controlled to dispense reagents from the first set of reagent sources to separate reaction sites simultaneously. The plurality of dispenser heads comprise a plurality of second dispenser heads, and flows through the second nozzles in the second dispenser heads are controlled to dispense reagents from the second set of reagent sources to separate reaction sites simultaneously.